William Held, PhD
“Epigenetic Modifications in Development and Cancer”
DNA methylation is intrinsically linked to chromatin structure and gene expression. Changes in DNA methylation during development suggest that it may play a role in development and in tissue-specific differentiation. Restriction Landmark Genomic Scanning (RLGS) is being used to identify sites of developmental and tissue specific differences in DNA methylation (Tissue Specific Differentially Methylated Regions, T-DMRs). RLGS is a method for the two dimensional display of end-labeled DNA restriction fragments. Using NotI as the restriction landmark, RLGS targets gene rich CpG island regions and detects differences in DNA methylation since cleavage by NotI is methylation sensitive. RLGS is being performed on a collection of different tissues from C57BL6/J mice of different ages to identify genomic sites of developmental and tissue specific DNA methylation (T-DMRs). New advances in development of “virtual” RLGS software makes it possible to rapidly clone “in silico” the genomic regions with tissue specific differences in DNA methylation/chromatin structure. Quantitative and semi-quantitative methylation sensitive PCR, along with bisulfite sequencing, is being used to delineate the boundaries and density of methylation in the regions. The tissue expression patterns of genes associated with these regions are accessed using available microarray data to evaluate possible “long distance effects” of chromatin structure on gene expression. Quantitative RTPCR methods are being used to evaluate expression of genes with apparent CpG island promoter DNA methylation. The results indicate that 5% or more of the CpG islands in the genome are T-DMRs disputing the general notion that all CpG islands are unmethylated. Some of the T-DMRs are within 5’ promoter CpG islands of genes, which exhibit a tissue specific expression pattern that is consistent with methylation status and a role in tissue differentiation. However, many T-DMRs are located in introns, 3’ exons, and in intergenic regions suggesting that DNA methylation may have novel and currently unknown effects on genome function.
It is becoming increasing clear that the genome is vulnerable to epigenetic alterations as well as genetic alterations that have consequences for promoting malignant growth. Aberrant epigenetic alterations are a hallmark of most cancers. Cancer cells tend to be globally hypomethylated but contain regions of CpG island hypermethylation. There is considerable evidence that indicates that DNA hypomethylation causes chromosomal instability possibly by altering chromatin structure in repetitive DNA and promoting DNA recombination events resulting in DNA deletions and rearrangements. In contrast, it is estimated that about 50% of the known tumor suppressor genes can be silenced by DNA hypermethylation as an alternative to mutation or DNA deletion. One aim of our research is to identify targets of CpG island methylation associated with human colon tumorigenesis and mouse model of tumorigenesis. Using NotI or other restriction enzymes that have GC rich recognition sequences as the restriction landmark, RLGS targets gene rich CpG island regions and primarily detects alterations in DNA methylation status although DNA deletions and amplifications can also be detected. RLGS loci exhibiting frequent alterations associated with human colon tumorigenesis are being cloned using in silico based virtual RLGS computational methods. Both Human tumors and corresponding mouse tumor models are being analyzed for common sites of aberrant methylation which would strongly suggest that the alteration impacts malignant growth. Array Comparative Genomic Hybridization (CGH) is being performed on the same tumors analyzed by RLGS to integrate epigenetic and genetic aberrations associated with human colon cancer.
This will allow us to establish whether there is a class of tumors with high epigenetic instability that can be distinguished from those with high genetic instability and whether there is cooperative interaction between epigenetic and genetic aberrations. The methylation status of selected RLGS loci is being evaluated by methylation sensitive PCR and bisulfite sequencing to determine the boundaries and density of DNA methylation in the region. Quantitative expression assays are being used to assess the expression of putative target genes in primary tumors to determine whether the CpG island alteration is associated with a change in gene expression. Results indicate that methylation in tumors may be associated with up-regulation as well as silencing of gene expression. The identification of novel targets of CpG island alteration may uncover new pathways of tumorigenesis and further our understanding of how epigenetic alterations impact on the malignant phenotype. These studies may also help to identify specific loci linked to age related methylation and tumor susceptibility. Finally, in the future, the novel targets may have broad application in defining patient populations that are more susceptible to cancer and possibly monitoring methylation changes with age or during administration of drugs for treatment or prevention.